This a continuation in part of application Ser. No. 08/974,737, filed Nov. 19, 1997, now allowed, which is a continuation of application Ser. No. 08/911,825, filed Aug. 15, 1997, now issued as U.S. Pat. No. 6,054,321, which is a continuation in part of application Ser. No. 08/706,408, filed Aug. 30, 1996, now allowed, which claims the benefit of the earlier filing date of a U.S. provisional patent application Ser. No. 60/024,050 filed on Aug. 16, 1996 each of which are herein incorporated by reference.
Fluorescent molecules are attractive as reporter molecules in many assay systems because of their high sensitivity and ease of quantification. Recently, fluorescent proteins have been the focus of much attention because they can be produced in vivo by biological systems, and can be used to trace intracellular events without the need to be introduced into the cell through microinjection or permeablization. The green fluorescent protein of Aequorea victoria is particularly interesting as a fluorescent protein. A cDNA for the protein has been cloned. (D. C. Prasher et al., “Primary structure of the Aequorea victoria green-fluorescent protein,” Gene (1992) 111:229-33.) Not only can the primary amino acid sequence of the protein be expressed from the cDNA, but the expressed protein can fluoresce. This indicates that the protein can undergo the cyclization and oxidation believed to be necessary for fluorescence. Aequorea green fluorescent protein (“GFP”) is a stable, proteolysis-resistant single chain of 238 residues and has two absorption maxima at around 395 and 475 nm. The relative amplitudes of these two peaks is sensitive to environmental factors (W. W. Ward. Bioluminescence and Chemiluminescence (M. A. DeLuca and W. D. McElroy, eds) Academic Press pp. 235-242 (1981); W. W. Ward & S. H. Bokman Biochemistry 21:4535-4540 (1982); W. W. Ward et al. Photochem. Photobiol. 35:803-808 (1982)) and illumination history (A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995)), presumably reflecting two or more ground states. Excitation at the primary absorption peak of 395 nm yields an emission maximum at 508 nm with a quantum yield of 0.72-0.85 (O. Shimomura and F. H. Johnson J. Cell. Comp. Physiol. 59:223 (1962); J. G. Morin and J. W. Hastings, J. Cell. Physiol. 77:313 (1971); H. Morise et al. Biochemistry 13:2656 (1974); W. W. Ward Photochem. Photobiol. Reviews (Smith, K. C. ed.) 4:1 (1979); A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995); D. C. Prasher Trends Genet. 11:320-323 (1995); M. Chalfie Photochem. Photobiol. 62:651-656 (1995); W. W. Ward. Bioluminescence and Chemiluminescence (M. A. DeLuca and W. D. McElroy, eds) Academic Press pp. 235-242 (1981); W. W. Ward & S. H. Bokman Biochemistry 21:4535-4540 (1982); W. W. Ward et al. Photochem. Photobiol. 35:803-808 (1982)). The fluorophore results from the autocatalytic cyclization of the polypeptide backbone between residues Ser65 and Gly67 and oxidation of the −β bond of Tyr66 (A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995); C. W. Cody et al. Biochemistry 32:1212-1218 (1993); R. Heim et al. Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994)). Mutation of Ser65 to Thr (S65T) simplifies the excitation spectrum to a single peak at 488 nm of enhanced amplitude (R. Heim et al. Nature 373:664-665 (1995)), which no longer gives signs of conformational isomers (A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995)).
Fluorescent proteins have been used as markers of gene expression, tracers of cell lineage and as fusion tags to monitor protein localization within living cells. (M. Chalfie et al., “Green fluorescent protein as a marker for gene expression,” Science 263:802-805; A. B. Cubitt et al., “Understanding, improving and using green fluorescent proteins,” TIBS 20, November 1995, pp. 448-455. U.S. Pat. No. 5,491,084, M. Chalfie and D. Prasher. Furthermore, engineered versions of Aequorea green fluorescent protein have been identified that exhibit altered fluorescence characteristics, including altered excitation and emission maxima, as well as excitation and emission spectra of different shapes. (R. Heim et al., “Wavelength mutations and posttranslational autoxidation of green fluorescent protein,” Proc. Natl. Acad. Sci. USA, (1994) 91:12501-04; R. Heim et al., “Improved green fluorescence,” Nature (1995) 373:663-665.)
A second class of applications rely on GFP as a specific indicator of some cellular property, and hence depend on the particular spectral characteristics of the variant employed. For recent reviews on GFP variants and their applications, see (Palm & Wlodawer, 1999; Tsien, 1998), and for a review volume on specialized applications, see (Sullivan & Kay, 1999). Biosensor applications include the use of differently colored GFPs for fluorescence resonance energy transfer (FRET) to monitor protein-protein interactions (Heim, 1999) or Ca2+ concentrations (Miyawaki et al., 1999), and receptor insertions within GFP surface loops to monitor ligand binding (Baird et al., 1999; Doi & Yanagawa, 1999).
The fluorescence emission of a number of variants is highly sensitive to the acidity of the environment (Elsliger et al., 1999; Wachter et al., 1998). Hence, one particularly successful application of green fluorescent protein (GFP) as a visual reporter in live cells has been the determination of organelle or cytosol pH (Kneen et al., 1998; Llopis et al., 1998; Miesenbock et al., 1998; Robey et al., 1998). The two chromophore charge states have been found to be relevant to the pH sensitivity of the intact protein, and have been characterized crystallographically in terms of conformational changes in the vicinity of the phenolic end (Elsliger et al., 1999), and spectroscopically using Raman studies (Bell et al., 2000). The neutral form of the chromophore, band A, absorbs around 400 nm in most variants, whereas the chromophore anion with the phenolic end deprotonated (band B) absorbs in the blue to green, depending on the particular mutations in the vicinity of the chromophore. WT GFP exhibits spectral characteristics that are consistent with two ground states characterized by a combination of bands A and B, the ratio of which is relatively invariant between pH 6 and 10 (Palm & Wlodawer, 1999; Ward et al., 1982). It has been suggested that an internal equilibrium exists where a proton is shared between the chromophore phenolate and the carboxylate of Glu222 over a broad range of pH (Brejc et al., 1997; Palm et al., 1997). Recent electrostatic calculations support this model (Scharnagl et al., 1999), and estimate the theoretical pKa for complete chromophore deprotonation to be about 13, consistent with the observation of a doubling of emission intensity at pH 11-12 (Bokman & Ward, 1981; Palm & Wlodawer, 1999).
In contrast to WT GFP, the chromophore of most variants titrates with a single pKa. The color emission and the chromophore pKa are strongly modulated by the protein surroundings (Llopis et al., 1998). G1u222 is completely conserved among GFP homologs (Matz et al., 1999), and its substitution by a glutamine has been shown to dramatically reduce efficiency of chromophore generation (Elsliger et al., 1999). Protonation of Glu222 in S65T and in GFPs containing the T203Y mutation (YFPs) is generally thought to be responsible for lowering the chromophore pKa from that of WT to about 5.9 in GFP S65T (Elsliger et al., 1999; Kneen et al., 1998), and 5.2-5.4 in YFP (GFP S65GN68L/S72A/T203Y) (Ormo et al., 1996; Wachter & Remington, 1999). In the YFPs, it is thought that the crystallographically identified stacking interaction of the chromophore with Tyr203 is largely responsible for the spectral red-shift (Wachter et al., 1998).
Unlike other variants, we have discovered that the YFP chromophore pKa shows a strong dependence on the concentration of certain small anions such as chloride (Wachter & Remington, 1999), and increases in pKa from about 5.2 to 7.0 in the presence of 140 mM NaCl (Elsliger et al., 1999). This sensitivity can be exploited to enable the creation of novel GFPs as biosensors to measure ions present both in the cytoplasm or in cellular compartments (Wachter & Remington, 1999) within living cells. The present invention includes the creation and use of novel GFP variants that permit the fluorescent measurement of a variety of ions, including halides such as chloride and iodide. These properties add variety and utility to the arsenal of biologically based fluorescent indicators. There is a need for engineered fluorescent proteins with varied fluorescent properties and with the ability to respond to ion concentrations via a change in fluorescence characteristics.